Bodily osmolarity is maintained in a narrow physiological range through the release of vasopressin (VP) and oxytocin (OT) into the blood where the hormones act to promote water retention and sodium excretion. The tachykinin, neurokinin B (NKB) and its NK3 receptor (NK3R) have a very unique relationship to VP because NKB and NK3R are co expressed by a majority of magnocellular VP neurons. Magnocellular NK3R are activated in response to physiological challenges because pretreatment with NK3R antagonists block both VP and OT release to both hyperosmolarity and hypovolemia/hypotension. Furthermore, NK3R play a traditional role in cellular signaling and immediate responses to hyperosmolarity and hypovolemia/hypotension and a more sustained role in regulating magnocellular neurons function through direct actions on gene transcription. Following a hyperosmotic challenge NK3R expressed by VP magnocellular neurons were shown to be translocated from the cell membrane to the cell nucleus by confocal microscopy, immuno-electron microscopy and Western blot. Nuclear NK3R was not detected under basal conditions. The translocation of activated g-protein coupled receptors into the nucleus has been hypothesized as a new paradigm for transcriptional regulation. Yet, in spite of the novelty and nuclear signaling importance of NK3R, we know little about NK3R signaling by magnocellular PVN neurons. The goals of this proposal are to identify the role of endogenous NKB and NK3R activation in magnocellular neuron function. Aim 1 will test the hypothesis that NK3R signaling is a "common path" for the activation of magnocellular neurons in the PVN in response to hyperosmotic and hypotensive challenges. Aim 2 will determine the time course of NK3R nuclear translocation and determine if NKB is translocated to the cell nucleus. The experimental hypothesis is that nuclear NK3R affects gene transcription through protein-protein interactions or by binding DNA. Co-immunoprecipitation and chromatin immunoprecipitation will be used to identify the nuclear targets of nuclear NK3R. Aim 3 will examine the effects of sustained hyperosmolarity on the regulation of NK3R and NKB mRNA in the PVN. Aim 4 will identify the source of release of the NK3R ligand (NKB). Results will identify NKB soma that are intrinsic to the PVN or that project to magnocellular PVN neurons and that are activated by hyperosmolarity. Collectively, the results will provide novel insights into membrane-bound receptors acting as nuclear transcription factors and the neurotransmitter systems controlling magnocellular neuron function. Dysfunction of VP release contributes to human pathophysiology, such as congestive heart failure, and the results may suggest novel treatment strategies and/or new insights into genomic underpinnings of human pathologies associated with NK3R system. PUBLIC HEALTH RELEVANCE The proposed research has the potential to contribute to therapies to deal with the dysregulation of neuroendocrine hormones that contribute to pathologies such as congestive heart failure and hypertension. The nuclear translocation of the neurokinin receptor to the nucleus is novel and has implications for a large number of pathologies, such as epilepsy, that are linked to this receptor system in the brain.